Sunday, August 28, 2011

Identification of bacteria within blood culture by Molecular Methods

Detection of bacteria with blood culture starts with testing if there are actually bacteria in blood or not.Only growth-positive cultures are used in subsequent analyses. Thereafter, pathogenic organisms are isolated using agar plates.Methods for identification of bacteria can be divided into two groups: hybridization based and amplification based.
Fluorescent in situ hybridization (FISH)
It is one of extensively used method for identification, visualization and localization of microorganisms in many fields. In brief FISH involves next steps. At first samples are fixed with aqueous solutions (paraformaldehyde for Gram-negative bacteria and 50% ethanol for Gram-positive bacteria) which are specific for Gram-negative and Gram-positive bacteria. Then bacterial samples are hybridized using fluorescently labeled probes that are complementary to the 16S rRNA in the cell.
Next steps include incubation and washing to remove unspecific probes. And finally samples can be visualizated with fluorescent or laser scanning microscopy (Barken et al., 2007)
At the present this technique allows identification within 2 hours of more than 95% of the bacteria usually found in blood. As long as this method needs species-specific probes, some bacteria can be identified only at the genus level. For example, detection of coagulase-negative staphylococci has problems with specific probe design (Oliveira at al., 2002)
Another methods are built upon identification using PCR.
Amplification based diagnostics are particularly helpful when the bacterial load is low or subculturing takes time. Moreover fast identification of bacteria associated with severe morbility and mortality is very helpful, because allows to rapidly detect most common bacterial infections (Peters at al., 2004)
PCR technique is important in molecular diagnostics of such bacterial infections in blood as Streptococcus pneumoniae and methicillin-resistance Staphylococcus aureus infections.
Using blood cultures is limited, since they are fastidious organisms, and PCR detection is more suitable. For example, there is a assay, which permits to idetify seven Streptococcus pneumoniae serotypes using multiplex PCR and has reliable results in dignostics (O’Halloran and Cafferkey, 2005)
Molecular methods based on polymerase chain reaction (PCR) technology have been developed for infection diagnosis and pathogen identification. These methods offer a new approach based on detection and recognition of pathogen DNA in the blood, or indeed other clinical samples, with the potential to obtain results in a much shorter time frame (hours) than is possible with conventional culture (Dark et al., 2009)
PCR based pathogen detection depends on the ability of the reaction to selectively amplify specific regions of DNA, allowing even minute amounts of pathogen DNA in clinical samples to be detected and analysed (Jordan JA et al., 2005)
The DNA sequence that is amplified is determined by the design of oligonucleotide primers, short pieces of synthesised DNA that bind to either end of the sequence and form the starting point for DNA replication by DNA polymerase.
For bacterial pathogens, two basic approaches have been taken in assay design, using either specific primers that detect a particular organism or, more commonly, universal primers that bind to conserved sequences in bacterial but not human DNA (Dark et al., 2009)
The latter approach has the potential to detect a large number of bacterial species in a given sample.
Assays that are limited to the detection of a specific organism have often been developed to address a specific clinical need (for example, rapid confirmation of the presence of meningococcoci in patients with meningitis) (Seward RJ et al., 2000)
In most cases, however, the detection of bloodstream infection requires assays capable of detecting a broad range of pathogens given that several microbial species may be involved, including infections with multiple organisms (Munson El et al., 2003)
Furthermore terminal restriction fragment length polymorphism (T-RFLP) and single strand conformation polymorphism (SSCP) analyses are a choice. They are generally used for typing of microorganisms from blood cultures.
SSCP method is based on species-unique SSCP patterns which now are determined only for 25 of usually meeting bacteria in blood culture (Turenne et al., 2000)
Whereas T-RFLP profiles for more bacteria were found by sizing fragments from restriction digests of PCR products deduced from two sets of 16S rDNA-specific fluorescent dye-labeled primers (Christensen et al., 2003)